Antifouling paints are used on a wide range of underwater structures in order to protect them from the development of fouling organisms. The leaching of the toxic substances from the matrix of the paint causes toxic effects not only to the fouling organisms but also on other “non-target” biota. The present study addresses the impact of the antifouling paint Flexgard VI-II on brine shrimp nauplii selected as convenient test organisms. The surface to volume (S/V) concept developed by Persoone and Castritsi-Catharios (1989) was used to determine S/V-LC50s for the test biota exposed to PVC test panels of 400–1000 mm2 surface coated with the antifouling paint in test vessels containing 20 ml seawater. Total ATPase and Mg2+-ATPase were also analyzed for coated surface areas inducing less than 50% mortality in the brine shrimp nauplii. The calculated S/V-LC50(24 h) was 24.6 mm2/ml, which shows the high toxic character of the antifouling paint. Decreased enzymatic activities were noted in the brine shrimp nauplii exposed to test panels of 50 and 100 mm2 in 20 ml seawater. The present study indicates that the “surface to volume” concept is an interesting methodology that can be applied with both lethal and sublethal effect criteria for the determination of toxic stress from leaches of painted surfaces.